Project Summary Defects in the long-range transport of membrane organelles and macromolecules underlie the pathogenesis and progression of many diseases including neurodegenerative disorders, mental retardation and traumatic brain injury. Long-range transport takes place along the microtubule (MT) cytoskeleton and is driven by the kinesin and dynein motors. Understanding the mechanisms of MT-dependent transport is essential for the development of therapies that aim to alleviate the neurotoxicity associated with transport blockages. Despite many advances, it remains largely unknown how long-range transport is spatially and temporally controlled. This is a fundamental gap in our knowledge of cell biology and impacts understanding of many fundamental processes including how neuronal proteins navigate a geometrically complex cytoskeleton and reach their proper destinations in axons and dendrites. Spatial regulation requires modulation of the activity of MT motors and their interaction with MTs. Notably, MTs are now thought to provide guidance cues through MT-associated proteins (MAPs) and tubulin modifications, affecting the itinerary of MT motors and their cargo. Knowledge of these MT-based guidance cues, however, is rather limited and the mechanisms underlying the spatial control of MT-dependent transport are little understood. The long-term goal of this project is to understand how MT- dependent transport is regulated by septins, a family of GTP-binding proteins that associate with MTs. Our preliminary data demonstrate that septins directly modulate MT-kinesin interactions, affecting the transport of kinesins and cargo in hippocampal neurons. Here, we will investigate this unprecedented function of septins, testing the hypothesis that septins comprise a novel regulatory module for the spatial guidance of membrane traffic. Based on work in progress, we will use cell biological and in vitro cell-free approaches to mechanistically determine: 1) how septins control neuronal membrane traffic, and 2) how the motility of kinesin motors is regulated by MT-associated septins. Our studies will provide new insights into the spatial regulation of MT-dependent transport and the neuronal functions of septins, which are abnormally expressed in neurological and psychiatric disorders. In the long-term, our studies will pave the way to new therapeutic strategies that aim to restore MT-dependent transport in diseased neurons.